Accurate segregation of duplicated chromosomes ensures that daughter cells get one and only one copy of each chromosome. Errors in chromosome segregation result in aneuploidy and have severe consequences on human health. Incorrect chromosome number and chromosomal instability are hallmarks of tumor cells. Hence, segregation errors are thought to be a major cause of tumorigenesis (Jallepalli and Lengauer, 2001;Yuen et al., 2005). A study of the mechanism of chromosome segregation is essential to understand the processes that can lead to errors. Tremendous progress has been made in recent years in identifying the proteins necessary for chromosome movement and segregation, but the mechanism and structure of critical force generating components and the molecular basis of centromere stiffness remain poorly understood. We have proposed a new model for the organization of pericentric DNA and cohesin in the centromere (Bloom et al., 2006). The key feature of the model is that the kinetochore promotes the organization of pericentric chromatin into a cruciform in mitosis such that centromere-flanking DNA is held together via intramolecular cohesion, while chromosome arms are paired inter-molecularly. The model provides a solution to the major paradoxes in the field and reconciles the organization of centromere DNA with the distribution of cohesin. Our experimental approaches combine model convolution microscopy, single chromosome tracking in living cells, and high resolution colocalization of kinetochore proteins. We have chosen budding yeast S. cerevisiae to characterize the force producing mechanisms and tension elements that reside at the interface of kinetochore-MT attachments. Only one microtubule (MT) attachment per kinetochore and the ability to engineer designer chromosomes for single-molecule studies makes budding yeast an ideal system. Our studies will test the hypothesis that the fundamental unit of segregation conserved throughout phylogeny is the budding yeast attachment site (kinetochore) that links the chromosome via synapsed pericentric chromatin to the microtubule.